Arrays of microparticles and methods of preparation thereof

ABSTRACT

This invention provides high unit density arrays of microparticles and methods of assembling such arrays. The microparticles in the arrays may be functionalized with chemical or biological entities specific to a given target analyte. The high unit density arrays of this invention are formed on chips which may be combined to form multichip arrays according to the methods described herein. The chips and/or multichip arrays of this invention are useful for chemical and biological assays.

This application is a continuation of U.S. application Ser. No.13/465,618, filed on May 7, 2012, which is a continuation of U.S.application Ser. No. 12/910,468, filed on Oct. 22, 2010, which is acontinuation of U.S. application Ser. No. 11/874,355, filed on Oct. 18,2007, which is a continuation of U.S. application Ser. No. 10/192,352(now U.S. Pat. No. 7,335,153), filed on Jul. 9, 2002, which claimspriority to U.S. Provisional Application 60/343,621 filed on Dec. 28,2001, the entire contents of each of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to high unit density arrays of microparticles andmethods of making same. This invention also relates to multichip arraysand their methods of manufacture. This invention further providesmethods for performing bioassays using high unit density arrays andmultichip arrays.

BACKGROUND OF THE INVENTION

An array format for biological and chemical analysis holds the promiseto rapidly provide accurate results while minimizing labor. [NatureGenetics, 1999 Vol. 21 (1) supplement pp. 3-4] Typically, arrays ofbiological probes such as DNA, RNA or protein molecules are formedeither by deposition and immobilization or by in-situ synthesis on inertsubstrates. In these prior art methods, array formation is usuallyaccomplished by attaching probe molecules directly to a substrate, whichmay be composed of organic materials (such as polymeric materials likenitrocellulose) or inorganic materials (such as glass or silicon).

The use of silicon as a substrate provides certain advantages related tothe well-established methods of semiconductor wafer and chip processing.In semiconductor processing, wafers are modified and transformed in aseries of multiple processing steps to create desirable features.Usually, a plurality of identical features are made on each wafersimultaneously by parallel processing to form individual segments on awafer. Dramatic savings in manufacturing time are achieved byfabricating identical features using parallel or batch processing. Inaddition, batch processing yields high chip uniformity, and by usingcertain photolithography and etching methods, very small (sub-micron)features can be precisely fabricated. Accordingly, structures with highfeature densities can be fabricated on a very small chip. Afterprocessing is completed, the individual segments are cut from wafers ina process known as singulation, to obtain a multiplicity of chips.[Peter Van Zant, “Microchip Fabrication”, 3^(rd) edition, McGraw-Hill1998].

Semiconductor wafers containing different functional chips can becombined either in final packaging processes by interconnectingdifferent chips or simply by bonding two wafers with differentfunctional chips, then cleaving the stack of wafers. The high efficiencyof the semiconductor fabrication process has significantly contributedto the rapid growth of the industry. Highly sophisticated systems havebeen developed for chip production, packaging, and quality control.

Biochips are arrays of different biomolecules (“probes”) capable ofbinding to specific targets which are bound to a solid support. Therehave been essentially two methods to prepare biochips.

The first method involves placing aliquots of solutions containingpre-synthesized probe molecules of interest on a planar substrate,followed by immobilizing the probe molecules in designated positions.For example, probe solutions can be dispensed (“spotted”) on a substrateto form a positionally encoded one-dimensional [Kricka, Larry J.,“Immunoassay”, Chapter 18, pages 389-404, Academic Press, 1996] ortwo-dimensional [U.S. Pat. Nos. 5,807,755 and 5,837,551] probe arrays ofcustomized composition. Molecular probes may be directly attached to asubstrate surface or may be attached to solid phase carriers, which inturn are deposited on, or attached to a substrate to form an array.Microparticles (“beads”) represent one type of such carrier. Beads offerthe advantage of separating the process of preparing and testingsubstrates from the process of preparing, applying and testing probe andassay chemistries [U.S. Pat. No. 6,251,691]. Beads of various sizes andcompositions have been extensively used in chemical and biochemicalanalysis as well as in combinatorial synthesis.

The deposition, printing and spotting methods for probe array productionhave several undesirable characteristics. First, even state-of-the-artdeposition and printing technologies only produce arrays of low featuredensity, reflecting typical spot dimensions of 100 microns andspot-to-spot separations of 300 microns. Second, methods of probedeposition described to date have failed to produce uniform spots, withsignificant spot-to-spot variations. Third, spotting methods, includingsuch variants as electrophoretic deposition to patterned electrodes[U.S. Pat. No. 5,605,662], require substantial instrumental andlogistical support to implement the production of arrays on anysignificant scale. In particular, spotting methods do not support batchfabrication of probe arrays. That is, while a batch processing formatmay be used to produce substrates efficiently, the subsequent step of“bio-functionalizing” these substrates by applying chemical orbiochemical probes is inefficient, because it does not conform to abatch format but instead requires many individual spotting steps. Thus,this process of manufacturing large numbers of identical functionalizedchips is far more time-consuming and expensive than a process that usesparallel processing procedures.

The second method of preparing probe arrays involves in-situphotochemical synthesis of linear probe molecules such asoligonucleotides and peptides using a process similar tophotolithography, a standard component of semiconductor processing.These methods have been most widely used in recent years to synthesize,in a parallel set of multi-step photochemical reactions, sets ofoligonucleotides in designated sections of glass or similar substrates[U.S. Pat. No. 5,143,854; Proc. Nat. Acad. Sci. USA, 1996, 93:13555-13560].

Although parallel processing to generate simultaneously a multitude ofprobe arrays directly on a wafer has the advantage of the scalabilityand intrinsic improvement in uniformity afforded by batch processing,serious drawbacks exist for the fabrication of probe arrays. First, onlysimple, relatively short linear molecules are suitably synthesized in aseries of single step reactions, and in practice, only arrays of shortoligonucleotides have been prepared by this method. Second, thereactions often do not proceed to completion, leading to significantcompositional heterogeneity. Third, all semiconductor processing must becompleted prior to the introduction of biomolecules, becausebiomolecules may not be compatible with the harsh environments incertain semiconductor processing steps. This limitation can preclude onefrom taking full advantage of the wide variety of semiconductorfabrication techniques. Fourth, if functionalization is performed in abatch fabrication format, that fabrication process defines the chemicalor biochemical composition (“content”) of each chip on the wafer. Thatis, to introduce a change in probe design requires that the entirefabrication process be changed accordingly.

Customization, while theoretically feasible, requires a change in thesequence of requisite masking steps required for photochemical synthesisof a desired set of probe molecules. The cost and time delays associatedwith this process renders customization infeasible in practice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the process of the invention.

FIG. 2 shows an example of a chip comprising a bead array. The chip iscomprised of three layers (L1, L2, L3). L1 is a silicon substrate with amicromachined array to accommodate beads (A1); L2, patterned SiO₂ (100nm thick); L3, is a layer of Si₃N₄ (510 nm thick).

FIG. 3 shows an example of wafer design.

FIG. 4 shows an example of the design of chips comprising bead arrays.The substrate is silicon (Si). The 12-tip star pattern is on a 100 nmthick layer of SiO₂. The area inside the star has no SiO₂ covering whilethe area outside the star has an SiO₂ covering. In the center, there isan array of closely packed hexagonal recesses. The total number of therecesses is 4012.

FIG. 5 provides examples of surface structures that can be used tosecure beads. H1 is a single-bead retaining hole with straightsidewalls. H2 is a pyramidal recess that can accommodate one bead. H3 isgroup of posts that confine one bead. Hx is a recess that can hold aplurality of beads.

FIG. 6 shows examples of array structures. A1 and A2 are arrays ofrectangular recesses. A3 is an array of hexagonal recesses.

FIG. 7 shows an example of chip grouping.

FIG. 8 illustrates the process of chip packaging. A, B, C, D are chipswith different functionalization. Wafers can be separated into chips bybreaking the wafer according to the scribing lines. Individual chipswith different functional groups which were separated from differentwafers can be placed together. A 4-chip package consists of fourdistinct functionalized chips bonded next to each other for biologicalapplication. The four chips can be arranged in a variety of ways, withnon-limiting examples including square or linear formats.

FIG. 9 illustrates a method of assembling chips by moving free chips inrows and columns.

FIG. 10 shows an example of a chip design that sets a probe array on acorner of each chip. By combining four such chips in the way shown inthe drawing, a larger array can be formed.

FIG. 11 illustrates the fabrication method for the chips comprising beadarrays of the invention.

FIG. 12 is a photograph of hydrogel formed on silicon wafer.

FIG. 13a is an illustration of the fluorescent images of a bead array ona chip before hydrogel formation and after gel peeling. The number ofbeads and bead positions were identical. FIG. 13b is an illustration ofon-chip reaction results with hydrogel treatment and without hydrogeltreatment.

FIG. 14 is an illustration of a mobile chip carrier and its applicationin conjunction with reaction chambers.

FIG. 15(a) illustrates an example of a random encoded array, FIG. 15(b)illustrates a library of chips. FIG. 15(c) illustrates a random assemblyof chips from the library of chips, FIG. 15(d) illustrates a randomtiling array.

FIG. 16 is an illustration of array design for simultaneous assemblingor sequential assembling of groups of beads with distinct sizes.

FIG. 17 is an illustration of multichip carrier design.

SUMMARY OF THE INVENTION

The present invention provides a parallel processing method that takesadvantage of semiconductor fabrication methods. In addition, the methodsin this invention are flexible enough to address different quantity anddifferent assay requirements. The invention combines the flexibility ofbeing able to select the array content with the high feature density andeconomies of scale afforded by parallel (batch) array assembly. Thisinvention provides a process for the assembly of random encoded, solidcarrier-displayed probe arrays of selectable composition in designatedpositions within delineated compartments on a substrate which may thenbe fractionated into a plurality of chips having arrays ofcarrier-displayed probes. In another embodiment, singulated chips(without solid carrier-displayed probes) derived from one or moresubstrates are contacted with a desired population of solidcarrier-displayed probes to form chips having the desired array. Theformation of multichip arrays is also provided by combining chipsprepared from different substrates having different populations ofcarrier-displayed probes. The invention also describes designs ofsubstrates and chips displaying solid phase carriers such as chemicallytagged microparticles so as to optimize the performance ofchip-displayed microparticle arrays in bioanalytical tests and assaysfor various target analytes including biomolecules such as nucleicacids, proteins, cells and the like.

The method for producing biochips according to this invention comprisespatterning a substrate to form a plurality of chip regions, delineatinga separating boundary between the chip regions, assembling at least onebead array comprising bio-functionalized, optically encoded beads on asurface of the substrate, and singulating the chip regions to formindividual biochips. As discussed above, singulation may be accomplishedprior to assembling a bead array on a chip surface. (In this context,the term “biochip,” as used herein refers to a chip having biomoleculesattached to its surface, e.g., for use in bioanalysis.) Non-limitingexamples of biomolecules include oligonucleotides, nucleic acidfragments, proteins, oligopeptides, ligands, receptors, antigens,antibodies, and individual members of biological binding pairs. Further,the term “singulate” or “singulation” as used herein refers to a processto obtain chips by breaking the connections between individual chipregions on a substrate or a subunit of a substrate containing more thanone chips. Also, the terms “functionalization” and“biofunctionalization” as used herein refer to a process to bindbiomolecules (e.g., molecular probes) to a substrate, includingattaching to bead surfaces.)

This invention also provides a method of making an assay devicecomprising a plurality of molecular probes. The method compriseschoosing a molecular probe from a probe library and affixing it to aplurality of beads to form a bead sub-population. The beadsub-population is affixed to a major surface of a substrate comprised ofchips possessing a decodable tag that identifies the wafer of origin.The wafer is then singulated to produce a plurality of biochips. Theprocess is repeated with at least one other bead sub-populationcomprising a different molecular probe. The resulting biochips are thenassembled to form a bioarray.

Another aspect of this invention are the assay devices preparedaccording to the method described above.

The devices of this invention include substrates that have beenpartitioned to define separable chip regions. Such substrates optionallymay comprise further patterning and partitioning to define subregionsfor restraining one or more solid carriers, e.g. beads.

In another embodiment, this invention comprises the partitioned andoptionally patterned substrates which further comprise one or morepopulations of solid carrier probes for detecting a target analyte.

The singulated chips formed by the fractionation of wafers describedabove, with or without the solid carrier-probe arrays is also anembodiment of this invention. Preferably, the chips comprise a solidcarrier-probe array.

This invention also includes assay devices for detecting one or moretarget analytes. Such assay devices of this invention comprise one ormore biochips comprising an array of functionalized beads suitable fordetecting one or more desired target analytes. In a preferredembodiment, a plurality of different biochips are affixed to a carrierto provide the ability to detect different target analytes.

Another aspect of this invention is to provide a method for performingbio-assays comprising contacting a plurality of biochips bonded to acarrier with a solution comprising at least one target analyte, anddetecting the analyte directly or indirectly. The plurality of biochipsmay comprise at least one sub-populations of biochips with abio-functionalized array. Optionally, the plurality of biochips maycomprise at least two sub-populations of biochips wherein the biochipsof the different sub-populations are different sizes or different beadarray geometries.

Yet another aspect of this invention is to provide a method ofperforming an assay using the assay devices described above. The methodcomprises exposing a biochip array of the assay device to a solutioncontaining at least one target analyte and detecting the reactionproducts.

Another aspect of this invention is to provide a method for fabricatinga carrier for biochips comprising covering a solid substrate that has atleast one hydrophilic major surface with a patterned hydrophobic layerthat is used to spatially define an array of biochips.

This invention also provides a process of assembling bead arrays on asurface of a semiconductor substrate comprising placing a patterneddielectric film on a surface of the semiconductor substrate, wherein thedielectric film forms boundaries on the substrate surface, and addingbeads in a solution to a region of the substrate designated for beadarrays, wherein the region is defined by the boundaries.

Another aspect of this invention is to provide a process for directlydepositing beads on a surface of a semiconductor substrate to form abead array, said process comprising adding a solution of beads to thesurface of a patterned semiconductor substrate containing structures forhousing the beads and mechanically agitating the solution to induce thebeads to settle in the structures.

Yet another aspect of this invention is to provide a bead arraycomprising a removable coating for protecting the bead array on abiochip. In this aspect, the bead array comprises a plurality of beadswith surfaces to which molecular probes are attached, and the coatinghas the property of being non-reactive towards the molecular probes onthe surfaces of the beads.

This invention also provides a method for quality control during thefabrication of a biochip. This method comprises optically encodingbio-functionalized beads, exposing a patterned substrate containingrecesses for housing beads to a solution containing the beads, andoptically imaging the beads to ensure that the recesses aresubstantially occupied.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides compositions and methods to design and producearrays of desired composition and layout comprising chemical orbiological entities, such as biomolecules like nucleic acids andproteins. Specifically, the methods of the invention described hereincombine the flexibility of real-time selection of array content and highfeature density with the economies of scale afforded by a parallelprocess for the assembly of a multiplicity of random encoded, solidcarrier-displayed probe arrays of selectable composition, in designatedpositions of delineated wafer compartments (“chips”). The invention alsoincludes methods for the formation of positionally and compositionallyencoded arrays of such chips. Further, the invention provides wafer andchip designs that optimize performance of solid phase carriers such astagged microparticles (“beads”) and tagged chips (“tiles”) inbioanalytical tests and assays involving biomolecules and cells.

This invention provides methods and processes for making high unitdensity arrays of microparticles which are biologically or chemicallyfunctionalized. Such arrays can be produced in adjustable quantities, ina flexible format and with pre-selected compositions. The methods andprocesses of the invention can be conducted in a batch and parallelformat. Specifically, the invention relates to the fabrication of sucharrays of microparticles on one or more wafers, such that a part or theentirety of a specific wafer displays one or more such microparticlearrays with a composition and functionality which can be pre-selected.The invention also relates to the packaging of the resulting array ofmicroparticles in a multichip format.

The present invention provides arrays with compositions that depend onthe end use of the array. Arrays containing from about one bead to manymillions can be made. Generally the array will comprise from one to asmany as a billion or more, depending on the size of the beads and thesubstrate, as well as the end use of the array. Preferred ranges forhigh feature density array are from about 1,000,000,000 (1 billion) to 1beads/mm², more preferably 1,000,000 to 100 beads/mm², most preferably100,000 to 1,000 beads/mm².

The microparticles of the invention are functionalized to includechemical or biological entities such as, for example, DNA, RNA andproteins. These entities can be selected depending on the application ofinterest thereby providing flexibility of selection of array content. Inaddition, since such an array of microparticles has a high featuredensity, it can be designed to optimize the array performance in thebioanalytical assay of interest. Examples of such assays are disclosedin PCT/US01/20179 and U.S. Pat. No. 6,251,691 which are all incorporatedherein by reference.

The methods of the overall process of the present invention can begrouped into four general categories, namely pre-assembly, assembly,post-assembly and packaging. Such grouping is not intended to limitcertain methods to certain groups. FIG. 1 is an illustration of oneembodiment of the process of the invention as further described below.

1. Pre-Assembly

The methods of pre-assembly include the implementation of chip layout,fabrication of the wafer based on such layout, optionally scribing ofthe wafer, followed by cleaning and inspection, if required. Singulation(as described below) may follow the methods of assembly or may followthe method of wafer fabrication (as shown in FIG. 1). In the event thatindividual chips are obtained from a wafer, the resulting chips aregrouped and each chip may be labeled as described below to identify suchchip based on its functionalization history.

1.1 Chip Layout

An example of chip layout is shown in FIG. 2. It is to be understoodthat a “chip” may be any three-dimensional shape. Each chip comprises asubstrate (e.g., layer 1 (L1)) where bio-functionalized beads can beassembled to form an array of microparticles. Many types of materialsmay be used as a substrate. Suitable materials have certain desirablecharacteristics. These characteristics can be classified as mechanical(e.g., strength), electrical (e.g., having an interfacial impedance thatcan be modified), optical (e.g., flatness, transparency, a well-definedoptical absorption spectrum, minimal auto-fluorescence, highreflectivity) and chemical (e.g., amenable to processes for definingprecise features or for depositing dielectric layers, surface reactivitythat permits covalent linkages). Non-limiting examples of suitablesubstrates include semiconductors (e.g., silicon), insulators (e.g.,sapphire, mica, and ruby), ceramic materials, and polymers (e.g., MYLAR™(polyester), KAPTON™ (polyimide), and LUCITE™ (acrylic)).

In certain embodiments, the substrate can be a semiconductor wafer, suchas single crystal semiconductor wafers which are commonly used in thesemiconductor device industry. In other embodiments, the substrate canbe any patternable solid substrate selected to be inert to the reagentsused in chip fabrication and bioassays. Non-limiting examples of suchsubstrates include glass, plastics, and polymers.

FIG. 2 is an illustration of a chip having a rectangular cross-sectionand is not intended to limit other chip geometries. In FIG. 2, L1 isrepresented as a middle layer (although the layers on both sides of L1are not required). The recess array A1 of L1 is where the bead array isto be built. The shape of the recesses A1 need not be square.Non-limiting examples of other suitable shapes include triangles,rectangles, pentagons, hexagons, and circles. One of the functions ofrecess array A1 is to help arrange and secure the beads by buildingregular structures on the wafer or chips to confine the movement ofbeads on the surface.

Optionally, the chip may also contain a second layer (L2). Layer L2comprises a patterned insulating dielectric layer (for example, silicondioxide). One example of a possible pattern is given by FIG. 3, whichshows a star-shaped pattern in the middle of the chip. The heavilyshaded area is the dielectric material and the white area is where thedielectric is removed. The thickness of the dielectric layer istypically, but is not limited to, 100 nm. If an electric field isapplied vertically through the chip, a non-uniform potential near thesurface of the chip due to the L2 pattern is formed. The electric fieldmay be applied to the surface in accordance with the process set forthin U.S. Pat. No. 6,251,691 incorporated herein by reference (such aprocess is referred to as “LEAPS”). Using LEAPS, beads in a liquidsolution that is applied to the surface of the substrate are subjectedto a change in the lateral electric field gradient when an AC potentialis applied to the substrate. This electric field gradient drives thebeads in the solution so that the beads will accumulate in area A1 wheresurface structures have been built on L1. Accordingly, the pattern of L2can be any pattern that may cause bead accumulation in a particular areaof the substrate, although it should be recognized that some patternsare more efficient at causing beads to accumulate than others.

Patterning of the dielectric layer L2 in accordance with apre-determined design facilitates the quasi-permanent modification ofthe electrical impedance of the electrolyte-insulator-semiconductor(EIS) structure formed by the bead solution-dielectric-semiconductor. Byspatially modulating the EIS impedance, electrode-patterning determinesthe ionic current in the vicinity of the electrode. Depending on thefrequency of the applied electric field, beads either seek out, oravoid, regions of high ionic current. Spatial patterning thereforeconveys explicit external control over the placement and shape of beadarrays.

Optionally, a chip comprising a bead array may contain a protectivepassivation layer, usually covering the surface. Layer L3 functions asan interface between the chip and liquid media, which can include thebead suspension, bioassay samples, or chip washing chemicals.Accordingly, layer L3 should be relatively robust against corrosion fromchemicals and the ambient environment. It also should protect thefunctional probes attached to the beads from electrostatic damage duringbead array assembly. In some embodiments, layer L3 also minimizes theadhesion of beads to the chip surface during bead array assembly. LayerL3 is preferably inert to biological samples and is preferablynon-fluorescent in the same wavelength range as that used forfluorescent detection in bioassays. In addition, its existence shouldnot create a change in the electric field distribution near the chipsurface which would prevent the use of LEAPS for bead assembling. By wayof example, the layer L3 may be a thin layer of LPCVD (low pressurechemical vapor deposited) silicon nitride with a thickness of from about40 to about 100 Å.

Layer L3 can also be engineered by chemical treatments to alter thesurface properties. For example, a silicon nitride surface could beoxidized to yield SiO_(x) (i.e., SiO₂ and/or substoichiometric siliconoxide) or silicon oxynitride (SiO_(x)N_(y)), both of which arehydrophilic and would facilitate dispensing aqueous samples. In otherembodiments, the surface SiO_(x), or SiO_(x)N_(y) can be furtherfunctionalized with silanol groups to yield a hydrophobic surface.

Finally, the backside of each chip can be coated with a metal or metalalloy for electric contact (preferably an ohmic contact). For example,if the chip is made from a silicon substrate, the backside of the chipcan be coated with a thin chromium adhesion layer and a thicker goldlayer, using routine processes in the semiconductor industry. Althoughthe gold coating is useful because it is inert to most chemicals and hashigh conductivity, other ohmic contact coatings can be used if they arechemically compatible with the other fabrication processes. Non-limitingexamples include titanium nitride/tungsten and titaniumtungsten/tungsten. The chips may be coated with a metal or metal alloybefore or after singulation.

Optionally, one side of a chip and/or its parallel opposite side can becoated with a magnetically responsive material. This can be achieved byassembling magnetic beads on either or both sides of the chip magneticbeads using routine assembly methods. The methods set forth in U.S. Ser.No. 10/032,657, filed Dec. 28, 2001 can be used, and are incorporatedherein by reference. The magnetically responsive material can be alsofunctionalized prior to assembly to provide additional chemical andbiological functionality. Alternatively, all sides of a chip can beencoded by randomly adsorbing beads on the chip carrier using methodsknown in the art. The configuration of the array provides a miniaturizedtag identifying the chip (“Chip ID”) as well as the wafer of origin(“Wafer ID”). Each ChipID is drawn from the number, S, ofdistinguishable configurations of a random encoded array of L positions,given by the number of ways in which n (unordered) samples of r (k)(indistinguishable) particles, 1≦k≦n, may be distributed among Lpositions:S(L;n;r(k),1≦k≦n)=L!/[r(1)!r(2)! . . . r(k)! . . . r(n)!]Illustrating the large number of possible combinations is the fact thatan array of L=16 positions, composed of n=4 distinguishable bead types,each type represented four times (r(1)= . . . r(4)=4) can display S(16;4; r(k)=4; 1≦k≦4)=16!/[(4!)(4!)(4!)(4!)], or approximately 63 milliondistinguishable configurations.

In using random encoded arrays to produce a number of tags T, whereT<<S, for many applications of practical interest, a large configurationspace of size S is sampled to reduce the chance for duplication. Aparticular advantage of constructing tags using random encoded beadarrays is the fact that, by the methods of the present invention, theyare readily produced, inexpensively, in miniaturized format and in largenumbers in a single process step. ChipID codes in the form of randomencoded bead arrays are readily constructed to share common subfields orsubcodes which can be used to determine whether two or more chipsoriginated from the same wafer. For example, if a total of n bead typesare used to produce ChipIDs for chips on N wafers, p types can bereserved, with p<n and p selected such that 2^(p)>N. Wafer-specificsubcodes containing only the remaining n-p bead types are thenconstructed. For example, given n=16 bead types to construct ChipIDscontaining a subcode identifying each chip to have originated in one ofN=100 wafers, p=7 bead types can be reserved to construct a binary codeof 7 digits to identify each of the 100 wafers by the absence of up toseven of the reserved bead types. For example, one of the wafers in theset will lack all 7 of the reserved types, another 7 will lack one ofthe reserved types. The encoding beads are functionalized and carryprobe molecules on their surface. The encoding magnetic particles canalso be magnetized and can exhibit chemical and biologicalfunctionality.

An example of a fabricated chip is shown in FIG. 4. The substrate is aSi(100), n-type phosphorus-doped wafer with a resistivity of 1.5-4ohm-cm. The chip is a square with 1.75 mm sides and a thickness of 0.5mm. Layer L2 is 1000 Å of thermally grown silicon dioxide with a 12-tipstar opening in the middle. The dimensions of the star are as indicatedin FIG. 4. In the center of the chip, there is an array of closelypacked hexagonal recesses comprising 68 rows and 59 columns. Thedimensions of the hexagonal recesses are as indicated in FIG. 4. LayerL3 is a 60 Å thick layer of LPCVD silicon nitride, which covers theentire chip except for the sidewalls and bottoms of the hexagonalrecesses, where there is only bare silicon with native silicon oxide.

FIG. 5 illustrates non-limiting examples of other structures suitablefor confining the movement of beads. H1 is a single-bead retainingrecess or cavity with straight sidewalls. H2 is an inverse pyramidalrecess that can accommodate one bead. H3 is group of posts that confineone bead. Hx is a recess that can hold a plurality of beads. The upperdrawings show a plan view of the structures, while the lower drawingsshow a cross sectional view. In one embodiment, a straight side wallcompartment H1 that accommodates only one bead can be used. Thisstructure is useful for confining beads in a liquid medium. The shadedarea in FIG. 5 is substrate material and the white area is empty space.The compartment shape is not limited to a square; for example apyramidal recess H2 may be used as a compartment to hold a single bead.Furthermore, although the bottoms of the recesses are preferably flat,they need not be in certain embodiments.

FIG. 6 shows examples of array structures. A1 and A2 are arrays ofrectangular recesses. A3 is an array of hexagonal recesses. A multitudeof structures can be fabricated on a chip or wafer to form an array or aplurality of arrays on the chip or wafer surface. The structures may beall identical or different types of structures and/or differently sizedstructures may co-exist. Three illustrative embodiments are illustratedin FIG. 6. In the drawings, the shaded areas are the recesses. Thenon-shaded areas are the original substrate surface (which may becovered with a thin film). Arrangement A1 is a regular Cartesian arrayof square recesses. Arrangement A2 is an alternating checkerboard arrayof square recesses. Arrangement A3 is an array of hexagonal recesses.Although the arrays of this invention are not limited to regular arrays,regular arrays are convenient for interpreting the reaction results.

The location of the array on a chip, of course, is not limited to thecenter. For example, the array can be situated on a corner such as thoseshown in FIG. 10. In addition there may be more than one array on anchip. For example, four arrays can be fabricated on a single chip asshown in FIG. 10. In some embodiments, different bead groups are addedto each of the four arrays on the chip. One method to accomplish thisbead distribution in a large-scale process is by using a mask that onlyexposes one array at a time on each chip. A distinct bead group is addedto the exposed arrays. The mask is then shifted to expose another arrayon each chip and the process is repeated. After repeating four times,each chip will have four arrays with four distinct groups of beads.

1.2 Wafer Fabrication

Several methods can be used to impart a selected chip layout on thewafer using wafer techniques, such as for example photolithography ormaterial etching. The methods selected depend on the wafer designrequirements. A wafer undergoes one or more fabrication cycles dependingon different requirements to yield a fully fabricated wafer. Eachfabrication cycle in this process comprises, but is not limited to,three steps: (i) material growth and/or deposition; (ii) lithography;and (iii) etching. Each cycle usually produces one structural layer.Depending on the target structures on the wafer, one or more layers maybe fabricated cycle by cycle.

For example, material growth or deposition can be accomplished by growthof SiO₂ on silicon, or chemical vapor deposition of dielectric materialsuch as SiO₂, Si₃N₄ or others, or deposition of metals such as aluminum,chromium, gold, titanium, or others. The lithography step can includephotolithography, e-beam lithography, x-ray lithography, or imprintlithography. The etching step can include the removal of a certainamount of material in certain areas defined by a masking layer, such as,but not limited to, a photoresist. Non-limiting examples of etchingmethods include anisotropic etching, such as reactive ion etching,crystal plane-biased wet chemical etching, or isotropic etching, such toisotropic wet chemical etching, vapor etching, or plasma etching.

1.3 Wafer Scribing

For the efficiency of functionalization, chip regions are delineatedafter the wafer fabrication process, so that the chip regions aresuitable for batch parallel processing. For this reason, the currentinvention prefers to scribe the wafer to delineate areas which will giverise following singulation to individual chips. In other embodiments ofthis invention, chips may be separated using techniques which do notrequire scribing. The purpose of the scribe lines is to produce lines ofbreakage to facilitate the separation of individual chips during thewafer singulation step without damaging or breaking the individual chip.It is to be noted that although the scribe line will cede to laterbreakage, they are sufficiently robust to enable the subsequent steps ofthe process without breakage of the chips. By way of example, scribelines can be produced using a wafer scribing machine (e.g. DISCO,Dynatex, or Loomis Industry) to create scribe lines that are only afraction of the thickness of the wafer. This is followed by theapplication of a roller in the direction perpendicular to the scribelines to further delineate individual chips. The wafer can be scribed byusing a diamond-tipped scriber; trenches between chips on a siliconwafer can be produced by chemical etching using wet chemicals, such aspotassium hydroxide/water solutions at elevated temperature, forexample. The wafer can also be dry etched by deep reactive ion etchingto yield well-defined trenches between the chips.

1.4 Wafer Cleaning and Inspection

During the step of wafer scribing, dust or particles may be generated.To protect the surface of the wafer, in one embodiment of thisinvention, a protective layer is applied to the wafer surface. Forexample, the layer can be in the form of an adhesive tape (if it doesnot damage the wafer surface), a photoresist coating, or some otherorganic coating. The protective layer is removed after scribing, bypeeling it off the wafer (for example if it is an adhesive tape) ordissolving it in an appropriate solvent. For example, a photoresistlayer can be removed by dissolving it in acetone and then rinsing thewafer with isopropyl alcohol. If trace amounts of the protective coatingmaterial are left on the wafer, more aggressive cleaning methods can beused. In one embodiment, the wafer is cleaned by an oxygen plasma toremove the trace amounts of organic material. In another embodiment, thewafer is cleaned by the RCA clean, a standard cleaning procedure in thesemiconductor industry which involves an ammonium hydroxide/hydrogenperoxide mixture that is heated to about 75° C. In another embodiment,the wafer is cleaned by a mixture of concentrated sulfuric acid andhydrogen peroxide at elevated temperatures (about 60° C.).

1.5 Chip Grouping

FIG. 7 shows an example of chip (C3) grouping. C1 is a wafer or anysubstrate unit that is convenient for batch fabrication such as thatused in the semiconductor industry; C2 is a sub-unit of C1 (could be awhole C1) which consists of a desired number of chips; C3 is a chip,which is the smallest unit of a biochip. Usually, C2 is an integratedunit for chip functionalization. After functionalization, C2 isseparated into individual C3's.

1.6 Bead Functionalization and Pooling

The terms “microsphere”, “microparticle”, “bead” and “particle” areherein used interchangeably. The composition of the beads includes, butis not limited to, plastics, ceramics, glass, polystyrene,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas sepharose, cellulose, nylon, cross-linked micelles and TEFLON™(polytetrafluoroethylene). (See “Microsphere Detection Guide” from BangsLaboratories, Fishers, Ind.) The particles need not be spherical and maybe porous. The bead sizes may range from nanometers (e.g., 100 nm) tomillimeters (e.g., 1 mm), with beads from about 0.2 micron to about 200microns being preferred, more preferably from about 0.5 to about 5micron being particularly preferred.

In some embodiments of this invention, the beads are functionalizedprior to being distributed on the wafer surface, such that each bead hasa specific type of biological probe linked on its surface. Variousmethods for functionalizing the beads are suitable for use with thisinvention. The appropriate method is determined in part by the nature ofthe material used to make the bead. For example, beads can befunctionalized by attaching binding agent molecules thereto, suchmolecules including nucleic acids, including DNA (oligonucleotides) orRNA fragments; peptides or proteins; aptamers and small organicmolecules in accordance processes known in the art, e.g., using one ofseveral coupling reactions known in the art (G. T. Hermanson,Bioconjugate Techniques (Academic Press, 1996); L. Illium, P. D. E.Jones, Methods in Enzymology 112, 67-84 (1985). In certain embodimentsof the invention, the functionalized beads have binding agent molecules(e.g., DNA, RNA or protein) covalently bound to the beads. Beads may bestored in a buffered bulk suspension until needed. Functionalizationtypically requires one-step or two-step reactions which may be performedin parallel using standard liquid handling robotics to covalently attachany of a number of desirable functionalities to designated beads. Beadsof core-shell architecture may be used, the shell composed in the formof a thin polymeric blocking layer whose preferred composition isselected; and functionalization performed in accordance with thetargeted assay application.

In some embodiments of this invention, the beads are color-coded withfluorescent dyes. For use in various assays, the beads may compriseadditional dye-tagged biological substances on their surfaces. To detectthe signal of the beads and assay, fluorescent microscopic imaging canbe used.

A bead library is established by preparing subpopulations of differentgroups of beads. Each bead subpopulation is prepared by affixing onetype of molecular probe from a probe library to a plurality of beads,forming the subpopulation. Each bead subpopulation is distinguishable bycolor coding with fluorescent dye or other method.

II. Assembly

Bead arrays are assembled by securing beads on the surface of a wafer orportion of wafer. Prior to securing the beads a wafer surface, a beadlibrary can be formed by chemical encoding or staining of beads withsets of optically distinguishable tags, such as those containing one ormore fluorophore dyes spectrally distinguishable by excitationwavelength, emission wavelength, excited-state lifetime or emissionintensity. The optically distinguishable tags made be used to stainbeads in specified ratios, as disclosed, for example, in Fulwyler, U.S.Pat. No. 4,717,655. Staining may also be accomplished by swelling ofparticles in accordance with methods known to those skilled in the art,(Molday, Dreyer, Rembaum & Yen, J. Mol Biol 64, 75-88 (1975); L. Bangs,“Uniform Latex Particles, Seragen Diagnostics, 1984]. For example, up totwelve distinguishable populations of beads can be encoded by swellingand bulk staining with two colors, each individually in four intensitylevels, and mixed in four nominal molar ratios. Combinatorial colorcodes for exterior and interior surfaces are disclosed in internationalApplication No. PCT/US/98/10719, which is incorporated herein byreference in its entirety. Color codes are also discussed in U.S. Pat.No. 6,327,410, which is hereby incorporated by reference in itsentirety.

There are many possible ways to secure beads on the surface of a chipwhen forming bead arrays. Recesses that are formed during waferfabrication steps provide compartments that retain the beads on thesurface of the substrate. The effectiveness of securing (orimmobilizing) the beads depends on the dimensions of the recess relativeto the size of the bead. Dimensions of recesses used for this purposeare such that the depth of the recess is about 0.5 to 1.5 times thediameter of the beads used. More preferably, the dimensions of therecess are such that when a bead is gravitationally stable in therecess, its highest point is below the top of the edges, and there isonly sufficient room to accommodate up to ⅓ the volume of another bead.In addition, while it is preferred that the size of the recess isgreater than the size of the bead, in this preferred embodiment, eachrecess should not be capable of accommodating more than one bead.Further, the openings of the recesses are slightly larger than thebeads. The hexagonal array shown in FIG. 4, for example, is compatiblewith beads having a diameter of 3.2 microns.

It may not be necessary to use recesses in a substrate to hold thebeads. For example, a plurality of posts can be arranged on a substratesurface to restrain the beads. A possible structure is shown in theupper drawing (plan view) and in the lower drawing (perspective view) ofFIG. 5c . In this case, each bead is confined by six posts around it.The number of posts is not limited to six, but could be three or more.Furthermore, any other raised or lowered surface structure, includingblocks, posts, bumps, and indentations may be used. In otherembodiments, large recesses capable of holding more than one bead may beused. For example, FIG. 5d shows a large recess with straight sidewalls. The overview in the upper drawing shows that the horizontaldimensions of the large recess are more than twice that of a beaddiameter.

As described above, the geometry and size of the recesses used in theassembly of micro-particle arrays can be varied. In certain embodiments,the geometry and size are varied by depositing a layer of silicon oxideor polymer after the holes are formed by etching. For example, recesseswith re-entrant sidewall profiles can be formed by this depositionprocess. In this context, the term “re-entrant sidewall profile” refersto the situation where the sidewall profile is such that the diameter ofthe recess opening at the surface is smaller than the diameter of therecess at its bottom. Recesses with re-entrant sidewall profiles formedby this method have a higher bead retention rate during processing andassaying.

Beads may be affixed to a surface by covalent bonds or by van der Waals,electrostatic, gravitational, magnetic or other forces. Combinations ofsuch bonding methods may also be used. In one embodiment, bead arrayscan be produced by picking aliquots of designated encoded beads fromindividual reservoirs in accordance with the specified arraycomposition. “Pooled” aliquots of suspension are dispensed onto selectedsubstrates such as a wafer delineated into compartments.

In other embodiments, the bead array can be prepared using LEAPS. Inthese embodiments, a first planar electrode that is substantiallyparallel to a second planar electrode (“sandwich” configuration) isprovided, with the two electrodes being separated by a gap containing anelectrolyte solution. The surface or the interior of the first planarelectrode is patterned by an interfacial patterning method, as describedbelow. Encoded and functionalized beads are introduced into the gap.When an AC voltage is applied to the gap, the beads form a randomencoded bead array on the first electrode (e.g., a chip or a wafer).Alternatively, an array of beads may be formed on a light-sensitiveelectrode e.g., chip or wafer) using LEAPS. Preferably, the sandwichconfiguration described above is also used with a planar light sensitiveelectrode and another planar electrode. Once again, the two electrodesare separated by a gap that contains an electrolyte solution. Thefunctionalized and encoded beads are introduced into the gap, and uponapplication of an AC voltage in combination with a light, they form anarray on the light-sensitive electrode.

Substrates (e.g., chips or wafers) used in the present invention may bepatterned in accordance with the interfacial patterning methods of LEAPSby, for example, patterned growth of oxide or other dielectric materialsto create a desired configuration of impedance gradients in the presenceof an applied AC electric field. Alternatively, a patterned substratemay be obtained by selectively doping interior regions of the substrate.Patterns may be designed so as to produce a desired configuration of ACfield-induced fluid flow and corresponding particle transport.Substrates may be patterned on a wafer scale by using semiconductorprocessing technology. In addition, substrates may be compartmentalizedby depositing a thin film of a UV-patternable, optically transparentpolymer that affixes a desired layout of fluidic conduits andcompartments to the substrate to confine a fluid in one or more discretecompartments, thereby accommodating multiple samples on a givensubstrate.

Spatial encoding, for example, can be accomplished within a single fluidphase in the course of array assembly by, for example, using LEAPS toassemble planar bead arrays in any desired configuration in response toalternating electric fields and/or in accordance with patterns of lightprojected onto the substrate. LEAPS creates lateral gradients in theimpedance of the interface between silicon chip and solution to modulatethe electrohydrodynamic forces that mediate array assembly. Electricalrequirements are modest: low AC voltages of typically less than 10V_(pp)are applied across a fluid gap of typically 100 μm between two planarelectrodes. This assembly process is rapid and it is opticallyprogrammable: arrays containing thousands of beads are formed withinseconds under an electric field. The formation of multiple subarrays,can also occur in multiple fluid phases maintained on acompartmentalized chip surface. Alternatively, spatial encoding isaccomplished by assembling separate chips, each carrying at least onerandom encoded array drawn from a specific pool, into designatedmultichip configurations.

In one embodiment, the process disclosed in PCT/US01/20179, incorporatedherein by reference in its entirety (the process referred to as “READ”),can be used to prepare custom bead arrays which can be used inperforming multiplexed biomolecular analysis according to the presentinvention. Using READ, the array can be prepared by employing separatebatch processes to produce application-specific substrates (e.g., chipat the wafer scale) and to produce beads that are chemically encoded andbiologically functionalized (e.g., at the scale of ˜10⁸ beads/100 μl ofsuspension). Preferably, the beads are subjected to respective qualitycontrol (QC) steps prior to array assembly, such as the determination ofmorphological and electrical characteristics, the examples of the latterincluding surface (“zeta”) potential and surface conductivity. Inaddition, actual assays are performed on beads in suspension before theyare introduced to the substrate. This is to optimize assay conditions,generally with the objective of maximizing assay sensitivity andspecificity and to minimize bead-to-bead variations. For substrates, QCsteps may include optical inspection, ellipsometry and electricaltransport measurements.

Once the chemically encoded and biologically functionalized beads arecombined with the substrate (e.g., chip or wafer), LEAPS or anotheractive deposition process described herein allows rapid assembly ofdense arrays on a designated area on the substrate. By assembling withinthe same fluidic phase, problems such as spot-to-spot or chip-to-chipvariability are avoided without the need for retooling or processredesign. Furthermore, the uniformity of these processes allow forchip-independent characterization of beads as well as optimization ofassay conditions. In addition, multiple bead arrays can be formedsimultaneously in discrete fluid compartments maintained on the samechip or wafer. Once formed, these multiple bead arrays may be used forconcurrent processing of multiple samples. The integration of LEAPS withmicrofluidics produces a self-contained, miniaturized, opticallyprogrammable platform for parallel protein and nucleic acid analysis.

Once the functionalized and encoded beads are prepared and then combinedwith the substrate, the binding interaction between the binding agent onthe beads and an analyte may be performed either before or after therandom encoded array is assembled on the substrate. For example, thebead array may be formed after the assay, subsequent to which an assayimage and a decoding image may be taken of the array. Alternatively, thebeads may be assembled in an array and immobilized by physical orchemical means to produce random encoded arrays. A DC voltage may beapplied to produce random encoded arrays. The DC voltage, typically setto 5-7 V (for beads in the range of 2-6 μm and for a gap size of 100-150μm) and applied for <30 s in “reverse bias” configuration so that ann-doped silicon substrate would form the anode, causes the array to becompressed, facilitating contact between adjacent beads within the arrayand simultaneously causes beads to be moved toward the region of highelectric field in immediate proximity of the electrode surface. Beadscan be anchored on the surface by van der Waals forces or “tethers”extending from the bead surface, e.g. polylysine and streptavidin.

After bead assembly, the chips or wafers are inspected and are imaged byfluorescent microscopy to obtain a decoding map. The decoding can belater used identifying the position and functionality of each individualbead.

The percentage of the array positions that are filled is preferablyhigher than 50%, more preferably higher than 90%. To test howeffectively the recesses retain the beads at the surface of thesubstrate, chips comprising bead arrays were placed in an aqueoussolution and were continuously shaken for three days. A comparison ofthe images taken before and after this test revealed that over 99% ofthe beads on all of the tested chips remained in the recesses.

III. Post-Assembly

During post-assembly, the bead arrays may be covered with a protectivesurface. Before or after covering the beads, the wafers comprising thebead arrays are singulated into one or more bead chips.

3.1 Securing of Microparticles

In certain embodiments of the invention, a gel covering the bead arrayarea can be used to prevent the beads from dislodging. In otherembodiments, chemical functional groups on the bottoms and/or thesidewalls of the recesses can be used to link the beads to the surface.A charged polymer can also be used to coat the chips prior to beaddeposition. The charge of the polymer coating is chosen to be oppositeto that of the beads, so that the beads will be electrostaticallyattracted to the polymer. When a bead is in a charged polymer-coatedrecess, the Coulombic attraction between the bead and the sidewalls andbottom of the recess serves to hold the bead in the recess. In this way,the bead retention rate during processing and assaying is increased. Insome embodiments, a second charged polymer is deposited on the chipsurface after beads have been placed in the recesses. The charge of thesecond polymer is chosen to be the same as that of the bead, so that nopolymer is deposited on the bead, but the surface charge on the chip isneutralized. Several variants of this techniques can be implemented withminimal alteration of the core process. For example, a singlepolyelectrolyte layer may be used, or a multi-layered structure (havingalternating positive and negative polymer layers) can be constructed toyield a coating with a more uniform and controlled thickness. Further,instead of polymers, charged polymer nanoparticles alone or incombination with charged polymers can also be used. An uncharged but lowT_(g) (glass transition temperature) polymer and/or nanoparticle coatingcan also be used to improve the adhesion of the beads to the chipsurface.

3.2 Protection of Assembled Arrays

Removable coatings can be used to protect the bio-functionalized beadsin the arrays of either a wafer before singulation into biochips or thesingulated biochips themselves. It is desirable therefore to have a wayto protect the bead array from ambient dust, dirt, and othercontaminants while the biochip or wafer is in storage. This inventionprovides protective coatings for biochips and wafers and methods ofpreparing such coatings. In preferred embodiments, the coatings protectthe beads in bead arrays from ambient contamination and prevent thedegradation of the bio-molecules (e.g. probes) on the surface of thebeads. Further, the coatings can be easily removed from the surface ofthe biochip prior to performing bioassays.

In certain embodiments, the coating comprises an inert, non-reducingsugar, such for example trehalose, which does not interact with reactivechemical moieties such as amino groups in peptides and proteins, andthus prevents the degradation or aggregation that is common when dryingwith other excipients.

In other embodiments, a hydrogel (e.g., an agarose hydrogel) may be usedto prevent contamination, dehydration, and physical damage duringstorage. Prior to performing a bioassay, the hydrogel may be peeled fromthe substrate surface. The act of peeling not only removes the hydrogel,but also cleans the surface of any extra beads that are not in arraypositions defined by recesses or other restraining structures. Theseextra beads, which are embedded in the hydrogel, can be recovered forfurther use.

3.3 Chip Singulation

After functionalization, the chip groups (wafer or subunit of a wafer)are singulated. If the wafer was previously scribed, it can besingulated by breaking the connections between the chips. This can bedone by rolling a roller on the back of the wafer in the directionperpendicular to the scribing lines, in accordance with the procedureoutlined in U.S. Pat. No. 3,790,051. Alternatively, singulation can beachieved by other methods such as using the GST Scriber/Breakermanufactured by DYNATEX INTERNATIONAL™. The individual chips obtainedthis way are ready for packaging.

In addition to the singulation methods described herein, any othermethod of singulation, such as for example laser cutting, can also beused to achieve the objectives of the invention.

In some embodiments, the wafer or subunit is singulated prior tobiofunctionalization. The individual chips can then bebio-functionalized identically or bio-functionalized by exposingsubpopulations of the chips to different bioactive groups.

IV. Packaging

Using the methods of the present invention, a multiplicity of chips canbe produced by the assembly of random encoded arrays of probe moleculesdisplayed on encoded beads. Each chip, cut from a uniquely identifiedwafer, may contain one or more random encoded bead arrays. It ispossible to generalize This method of random assembly according to theinvention encompasses an embodiment wherein the bead-displayed probes onchips containing random encoded arrays are members of large probelibraries that are displayed on tagged chips selected from amultiplicity of wafers. Chips from different wafers may be selected andassembled to form pooled chip sets. Preferably, chips display adecodable tag identifying the wafer of origin. Arrays of encoded chipsmay be formed by random assembly on a planar surface in a process alsoreferred herein as random tiling, illustrated in FIG. 15d . Randomtiling refers to a process of assembling a set of encoded chips into aplanar arrangement or array so as to permit optical inspection of eachchip or part of each chip within the assembly or array.

This hierarchy of scales for random assembly, from the bead array levelto the chip array level provides flexibility for quickly creating arraysof large probe sets of customized composition and high feature density.Such arrays could be used for displaying a large set of probes for geneexpression profiling, or for profiling the methylation of DNA by assaymethods known in the art. In addition, when it is desirable to exposemultiple probe arrays to separate reactions, a random tiling process ofaffixing a plurality of chips on a single support as discussed below,provides a rapid and flexible novel approach to implement pooling anddeconvolution strategies known in the art For example, arrays displayingpartially overlapping probe sets are readily produced by suitableconstruction and selection of chips.

Following the assembly of the bead arrays of this invention, wafers aresingulated to permit manipulation of individual tagged chips. In randomtiling, tagged chips that are selected from one or more wafers areplaced onto a surface (preferably provided by a flat substrate) on whichchips can be moved about to form a multi-chip assembly corresponding toa desired layout. To facilitate close packing, chips may be designed todisplay a convex symmetric shape such as a square, triangle or hexagon.To decrease the distance between bead arrays on adjacent chips, chipsmay be designed to display interlocking shapes (FIG. 14c ).

Sliding Assembly

In this embodiment, multiple singulated wafers are placed onto a commonlarge substrate with the sides displaying the probe arrays facing down.In preferred embodiments, the probe arrays are recessed to preventdirect contact with the substrate. One or more chips are randomlyselected from each wafer and, in a manner similar to sliding coins on atable top, arranged to form a chip array by sliding the chips into adesignated assembly area. This process may be generalized torow-and-column manipulation shown in FIG. 8. In this embodiment, thetiling process can be monitored and recorded by standard optical andmachine vision instrumentation available for semiconductor inspection.This instrumentation can be used to track chips from their respectivewafers of origin to their respective final positions, permitting directpositional encoding and decoding of the assembled chip array. Followingcompletion of the assembly process, a multi-chip carrier (as describedherein) is aligned with one or more chip arrays arranged in the assemblyarea and then lowered and bonded to the chips to form a multichipassembly. To facilitate bonding, carriers may be pre-coated withadhesive or may be coated with magnetic materials, if the chips arerendered magnetizable by methods described herein.

This method of sliding assembly preferably uses a mechanical tip, suchas a suction device capable of lifting and handling individual chips asknown in the an. Alternatively, magnetizable chips are manipulated usinga magnetic stylus capable of selecting one or more chips from eachwafer. Wafers (and chips contained thereon) may be rendered magnetizableby the deposition of a magnetic material such as nickel or a nickel-ironalloy (“permalloy”) by electroplating or electroless deposition, asunderstood in the art for example for semiconductors and ceramics.Alternatively, paramagnetic microparticles may be introduced, either asa part of the random encoded arrays of microparticles displaying probemolecules or as a separate feature, for example in the form of an arrayassembled in a designated portion of each chip. The array ofmagnetizable particles may be on the side of the chip containing therandom encoded probe array or on the opposite side.

Sliding assembly generally involves handling of individual chips andbecomes increasingly cumbersome as the number of constituent chips in achip array increases. This situation is exacerbated if the chips aresmall, displaying for example, linear dimensions of 100 μm or less. Forexample, small chips of cubic or near-cubic shape may be formed in thisdimension from ceramic substrates. In these situations, the individualchips are best handled by methods known in the art for the handling ofglass or polymer microparticles of similar dimensions.

Collective Assembly

In this embodiment, chips that are cut from individual wafers are storedin bulk suspension using an inert storage buffer such as high puritywater containing a trace amount of azide. The chips are suspended bymechanical or magnetic agitation. Pools of chips are formed bydispensing and mixings aliquots of selected suspensions. Optionally, atrace amount of glucose or other high soluble, molecular weightingredient may be added to this suspension to increase viscosity andthereby improve the flow characteristics. The suspension is thendeposited on a planar substrate either by spotting discrete aliquotsusing a syringe, pipette or capillary to achieve random deposition or byusing continuous methods known in the art to produce arrays of colloidalparticles including those invoking the action of flow and capillaryforces [Adachi, E., et al, Langmuir, Vol. 11 1057-1060 (1995); Science,Vol. 276, 233-235 (1997)].

In the case of random deposition, a template can be provided on thesubstrate to guide the placement of individual chips and to contain themin designated positions on the substrate. In one embodiment, chips maybe collected from the mixed suspension by inserting a mesh into thesuspension and retracting it, such that the individual chips areliterally lifted or “scooped” out. Preferably, chips, particularly whenplaced on a flat, feature-free substrate, are separated from one anothersufficiently so as to prevent partial overlap and stacking before theyare “racked up” into a close packing configuration. Separation isachieved, for example, by sliding assembly (see above) or by mechanicalagitation that takes advantage of inducing “drum modes” on flexiblesubstrates such as polymeric substrates, as practiced in the art.

In a preferred embodiment, chips are “racked up” by mechanical means,for example by entraining chips in a fluid flow directed parallel to thesubstrate surface in a sandwich flow cell in which chips forced againsta barrier at the far end of the flow cell.

Preferably, chips within a random assembly are oriented so as to exposethe active side displaying random encoded probe arrays. In cases wherethe active side is not exposed, the chips must be inverted. Inversion ofchips into the preferred orientation is achieved by cycles of mechanicalagitation and bonding of correctly oriented chips (coated with a heat orlight-activated bonding adhesive). Alternatively, inversion duringmechanical agitation is aided by displacing the center of mass towardthe undesired side of the chip, for example by metallization.Magnetizable chips can be deposited in the presence of magnetic fieldgradient aligned perpendicularly to the substrate surface, providing forsufficiently slow settling in a high viscosity medium to permit chips toadopt the correct orientation as they approach the surface.

Inversion also is facilitated by producing three-dimensional shapes,such as a pyramidal shape, the tip of the pyramid facing away from theactive surface, as produced by standard semiconductor etching methods.

The chips can be packaged in single or multi-chip packages. In amulti-chip package, chips containing different bioprobe arrays areplaced on the same carrier. FIG. 8 shows four chips packaged together toform a square combo chip or a linear combo chip. The four chips can beglue-bonded to a common carrier such as a glass slide, or they can beattached to a carrier by other methods, such as bonding magneticmaterials on the back of the chips so that they stick to a magneticcarrier. The chip handling is not limited to using pick-and-placeequipment. The chips can be grouped in rows or columns aftersingulation. These rows and columns of chips can be moved by confinementbars. FIG. 9 shows that by selectively arranging different chip rows,different combinations of chips can be obtained.

Another package design is shown in FIG. 10. Four chips with arrays onthe corners can be combined to form a chip with a larger array in themiddle. If the arrays on the four chips comprise distinct functionalprobes, the big array will contain four times as much information as asingle chip.

A multiplicity of chips can be produced by the assembly of randomencoded arrays of probes that are displayed on beads. Each chip mayinclude one or more random encoded bead arrays and may be cut from auniquely identified wafer. In another embodiment, chips containingrandom encoded arrays of probes on beads can be members of probelibraries. In this embodiment, each chip in a multichip array displays adecodable tag that identifies the wafer of origin.

A glass surface in the form of a slide or other similar surface may beused to make a multichip carrier. To prepare the slide as a carrier, acoating, such as TEFLON™ (polytetrafluoroethylene), may be applied insuch a manner as to leave circular openings or wells (i.e., areas ofglass without any TEFLON™ covering). Each well is a circle with 6.5 mmin diameter. One or more chips can be bonded to the glass surface withina well. A typical glass slide is 25×75 mm and 1 mm thick, with a 2×5array of wells. With typical chip sizes of 1.75×1.75 mm, up to fourchips can be bonded to the glass surface in each well. Each chip in thesame well can have distinct bead groups that were assembled prior tobonding to the carrier. For example, if each chip has an arraycontaining 39 types of bead groups, a well with 4 distinct chips wouldhave a total of 4×39=156 types of beads. On the other hand, for largerchips (e.g., a 4.5×4 5 mm square) an entire well is occupied by a singlechip. For the well dimensions described herein, each well can hold up to40 μl of liquid (usually an aqueous solution). Typically, a 20 μl volumeof sample solution is added to each well for biological reactions, suchthat each chip is totally covered by the sample solution. Because theTEFLON™ (polytetrafluoroethylene) coating outside the wells ishydrophobic, the aqueous samples do not spill out. The format of acarrier slide can be designed to fit certain applications. For example,a single row of 8 wells on a slide can be used to analyze 8 samples.Furthermore, a 4×8 array of wells can be used to analyze 32 samples.Similarly, more wells (e.g., 96, 384, and 1536) can be arranged on asingle slide to analyze more samples.

In certain embodiments of a mobile chip carrier, chips are bonded to asubstrate such as glass, stainless steel, plastic materials, silicon, orceramic materials. The whole carrier unit is movable and can betransported during processing to expose the chips to different reactionmedia, such as reaction chambers, washing chambers, and signal readingstages.

In other embodiments, the mobile chip carrier comprises a chamber orchambers in which the chips are secured. By housing the chips inside themobile chip carrier, contamination during transport can be minimized. Incertain embodiments, the chamber or chambers of the mobile chip carrieralso serve as a processing environment. Reactive gases or liquidsolutions for various purposes, such as performing a bioassay orcleaning the chips, may be admitted into the chamber and subsequentlyevacuated, if desired. Additionally, the mobile chip carrier may possessmeans for changing the thermodynamic properties of the chamber, such asthe chamber pressure or temperature.

V. Assays

The biochips of the invention comprising bead arrays are useful forconducting various bioanalytical and chemical assays. Once assembled,the bead arrays on the biochips of the invention may be imaged to recordassay signals and may be decoded to identify target analytes bound tothe probes associated with individual beads within the array. The beadarray provides a system which can be used to read the results ofmultistep bioanalytical or chemical assay sequences. In addition,multiple target analytes are capable of being detected simultaneouslydue to the presence of a plurality of probes directed to differenttarget analytes comprising the arrays. Besides providing the ability todetect the presence or absence of specific target analytes, the beadarrays of the invention also find applicability in the determination ofaffinity constants for the target analytes which bind to the probes.Thus, the biochips have broad applicability to detect for example,biomolecules such TNF-alpha and Il-6. Other non-limiting applicationsinclude genotyping by polymorphism analysis; gene expression analysis;quantitative multiplexed profiling of cytokine expression, analysis ofgenes and gene products within the same fluid sample; affinityfingerprinting; and multiplexed analysis of reaction kinetics. Otherassays and analytical determinations, such as those referred to in U.S.Pat. No. 6,327,410, which is incorporated herein by reference, may beadapted for use with the biochips of this invention.

EXAMPLES

The present invention will be better understood from the Examples whichfollow. However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention described in the claims which follow thereafter.

Example 1 Wafer Fabrication and Design of Chips Comprising Bead Arrays

The fabrication process of the chip comprising a bead array, as shown inFIG. 4, is described in FIG. 11. The substrate was a 100 mm diameter,0.5 mm thick, silicon wafer with crystal orientation of (100), n-typephosphorus doped. A suitable resistivity range for these wafers is 1.5-4Ω-cm. Wafers were usually fabricated in batches of up to 25. The firststep comprises SiO₂ growth. The wafers were first cleaned by the RCAcleaning process, which comprises the steps of (1) soaking the wafers ina mixture of NH₄OH:H₂O₂ (30%):H₂O in a volume ratio of 1:1:5 at 75° C.for 10 minutes; (2) rinsing with a cascade water batch cleaning using 18Me-cm water; (3) soaking the wafers in a mixture of HCl (36%):H₂O₂(30%):H₂O in a volume ratio of 1:1:5 at 75° C. for 10 minutes; and (4)rinsing with a cascade flow water batch cleaning until the water in thebath is at least 16 MΩ-cm. The wafers were spun dry before being placedin a horizontal furnace for SiO₂ growth. The wafers were placedvertically on a quartz boat and introduced to an oxidation furnace at1050° C. which had O₂+HCl(4%) at a pressure of 760 torr. The oxidationtime was 34 minutes. A uniform 1000 Å SiO₂ layer was obtained by thismethod, as verified using ellipsometiy (refractive index: n=1.46,thickness variation: <5%).

The wafers with SiO₂ were spin-coated with photoresist (Shipley 1813) ata spin rate of 4000 rpm (spin time 30 seconds), then baked on a hotplate at 115° C. for 60 seconds to remove the solvent. The wafer wasthen exposed to UV light (365-405 nm) in a contact lithography stepwhich used Hybrid Technology Group's (HTG) system 3HR contact/proximitymask aligner. Following UV exposure, the wafer was developed by AZ300MIF developer for 60 seconds, rinsed in DI water and blown dry with astream of compressed dry nitrogen. The wafers were then submerged inbuffered oxide etchant (6:1 mixture of ammonium fluoride and 50% aqueoushydrogen fluoride) for 2 minutes to etch away the SiO₂ on the exposedarea (the star in FIG. 11). The wafers were subsequently rinsed with DIwater, then soaked in 1165 Microposit photoresist remover at 60° C. for60 minutes to remove the photoresist. The wafers were then rinsed in DIwater and blown dry with the jet of dry compressed nitrogen. This entireprocedure results in wafers with a patterned oxide layer.

Following the oxide patterning step, the wafers were cleaned by the RCAprocess, and then placed in a horizontal furnace for silicon nitride(Si₃N₄) deposition. Two types of silicon nitride can be used: standardand low stress nitride. The conditions for deposition are as follows:LPCVD nitride (standard), pressure=200 mtorr, temp=800° C., SiCl₂H₂=30sccm, NH₃=90 sccm; LPCVD nitride (low stress): pressure=150 mtorr,temp=850° C., SiCl₂H₂=47 sccm, NH₃=10 sccm. After 2 to 3 minutes ofdeposition, the Si₃N₄ film thickness is between 60-90 Å.

The next step is to fabricate the array structures. The wafers werespin-coated with photoresist OCG 12i at a spin rate of 4000 rpm (spintime 30 seconds) and then baked on a hot plate at 90° C. for 60 secondsto remove solvent. The wafers were exposed to UV light (365 nm) andrepeat lithography was performed using a GCA-6300 10× i-line Stepper.After exposure, the wafer was baked on a hotplate at 115° C. for 90seconds before being developed by AZ300 MIF developer for 60 seconds,rinsed in DI water, and blown dry with stream of compressed drynitrogen. The wafers were baked in a 90° C. oven for 20 minutes. Thewafers were then etched in a Plasma Therm 72 etcher to remove thesilicon nitride in the exposed area (the hexagonal features in thearrays) using CF₄ gas reactive ion etching. Oxygen reactive ion etchingwas then used to remove residual polymeric material on the hexagonalfeatures. The hexagonal recesses were fabricated by Deep Reactive IonEtching (DR1E) using a Unaxis SLR 770 ICP Deep Silicon Etcher (licensedBosch fluorine process). The process was adjusted so that it takes 2-3minutes to etch 3.8 micron-deep recesses. The control of the depth iswithin 0.3 microns. After etching, the wafers were soaked in 1165Microposit photoresist remover at 60° C. for 60 minutes to remove thephotoresist. The wafers were rinsed in DI water and blown dry with a jetof dry compressed nitrogen. The wafers were then processed in aGaSonics, Aura 1000, Downstream Photoresist Stripper by oxygen plasmafor 90 seconds to remove any residual polymer inside the hexagonalrecesses generated during the DRIE process. The wafers were thenspin-coated with a protective photoresist coating (Shipley 1813, spinrate of 4000 rpm, spin time 30 seconds), then baked on a hot plate at115° C. for 60 seconds to remove solvent. The wafers were sent out to acommercial vendor for backside coating of 500 Å of gold with 100 Å ofchromium as the adhesion layer. The backsides of the wafers were shippedof the native silicon oxide layer immediately prior to the coatingprocess using argon ion sputtering.

The fabricated wafers were saw cut on the surface to define each chip(dimensions of each chip, 1.75×1.75 mm square). The depth of the cutswere ⅔ of the thickness of the wafers. After saw cutting, the waferswere cleaned by soaking them in 1165 Microposit photoresist remover at60° C. for 60 minutes to remove the photoresist, then rinsing in DIwater and blowing dry with a stream of compressed dry nitrogen. Usuallythe wafers were then soaked in NanoStrip (a mixture of concentratedsulfuric acid and hydrogen peroxide) at 60° C. for 2 hours, then rinsedin DI water and blown dry with a stream of compressed dry nitrogen.After these procedures, the wafers are ready for the bead assembly step.

After bead assembly, the extra beads that are not secured in recessesmay be removed. One method for removing unsecured beads is to wipe thechip or wafer surface with moistened cotton applicators. Another methodis to wash away the unsecured beads using water jets nearly parallel tothe chip or wafer surface. Yet another method comprises growing a gel onthe surface and subsequently peeling off the gel.

Example 2 Functionalization of Beads and Formation of a Bead Array

Color encoded, tosyl-functionalized beads of 3.2 μm diameter were usedas solid phase carriers. Several sets of distinguishable color codeswere generated by staining particles using standard methods (Bangs. L.B., “Uniform Latex Particles”, Seragen Diagnostics Inc., p. 40). Stainedbeads were functionalized with Neutravidin (Pierce, Rockford, Ill.), abiotin binding protein, to mediate immobilization of biotinylated probesor primers. In a typical small-scale coupling reaction, 200 μl ofsuspension containing 1% beads were washed three times with 500 μl of100 mM phosphate buffer/pH 7.4 (buffer A) and resuspended in 500 μl ofthat buffer. After applying 20 μl of 5 mg/ml neutravidin to the beadsuspension, the reaction was allowed to proceed overnight at 37° C.Coupled beads (i.e., beads with bio-functional molecules attachedthereto) were then washed once with 500 μl of PBS/pH 7.4 with 10 mg/mlBSA (buffer B), resuspended in 500 μl of that buffer and reacted for 1hour at 37° C. to block unreacted sites on bead surface. After blocking,beads were washed three times with buffer B and stored in 200 μl of thatbuffer.

Probes (for the detection of target molecules) and primers (which can beused as templates to extend the hybridized DNA target for subsequentcatalytic reactions to identify the reacted probes) that were to becoupled to the beads were biotinylated at the 5′ end; a 15-carbontriethylene glycol linker was inserted between biotin and theoligonucleotide to minimize disruptive effects of the surfaceimmobilization on the subsequent reactions. For each primer, a bindingreaction was performed using 50 μl of bead suspension. Beads were washedonce with 500 μl of 20 mM Tris/pH 7.4, 0.5M NaCl (buffer C) andresuspended in 300 μl of that buffer. A primer solution (2.5 μl of a 100μM solution) was added to the bead suspension and allowed to react for30 minutes at room temperature. Beads were then washed three times with20 mM Tris/pH7.4, 150 mM NaCl, 0.01% triton and stored in 20 mM Tris/pH7.4, 150 mM NaCl.

An exemplary bead array was assembled as follows. The bead suspensionobtained through the procedures described above was washed withde-ionized water (All water used was highly purified and sterilized witha resistivity of 18 MΩ-cm or higher) five times before being suspendedin 0.01 mM of TRIS Base+0.01% Triton x-100 water solution. The beadcontent of the suspension was 0.5%. Two microliters of the beadsuspension was added to the surface of a 4.5 mm square chip comprising abead array by micro-pipet. The chip was then subjected to the process ofLEAPS. The counter electrode was a piece of glass coated by a layer ofindium tin oxide (ITO). The gap between the surface of the chip and theITO-coated glass was 100 microns. AC power was applied in the sequencelisted in Table 1.

TABLE 1 AC supply sequence. The voltage is half of the peak-to-peakamplitude. Time Frequency Voltage Step (minutes) (Hz) (±volts) Function1 2 2000 3 AC on 2 2 1000 3 AC on 3 2 500 3 AC on 4 2 2000 3 AC on 5 2500 3 AC on 6 2 2000 3 AC on 7 2 200 3 AC on 8 2 2000 3 AC on 9 2 200 3AC on 10 0 200 0 AC stop

After the sequence was completed, the beads within the area spanned bythe star shaped pattern were concentrated in the array area. The flowpattern induced by the presence of the star-shaped pattern helps toconcentrate the beads. After waiting 15 minutes for the beads to settle,the device was slowly soaked in pure water. The ITO glass coating wasslowly lifted, and the water was slowly drained so that the chip surfaceemerged. At this point, the surface could be dried by either leaving thechip at room temperature for an extended period or by baking the chip inan oven at 55° C. for 5 minutes. The dried chip was soaked in pure waterfor 15 minutes, and then the chip surface was gently wiped with a wetcotton swab several times to remove the beads that were not in thearray. The chip was subsequently rinsed with pure water three timesbefore being dried by blowing compressed nitrogen on its surfaces.

Finally, the chip was inspected by fluorescence light microscopy toensure that no extra beads were outside the array.

Example 3 Forming a Bead Array

A bead slurry was directly dispensed onto the array area on a chip. Awet cotton applicator (K1) was used to gently stir the bead slurry onthe array surface. The motion of K1 can be circular, linear or someother meaningful mode, and is usually parallel to the chip surface.After stirring the slurry several times, the beads were moved into thearray. Then, the chip surface was cleaned by using K1 to wipe away extrabeads that were not in the array. This process can be scaled up fromsingle chips to wafer-scale multi-chip assembly, and can be automated.

An example of a processing procedure for forming bead arrays is asfollows. Two microliters of 1% microparticles (approximately 3.2micrometers in diameter) in 100 microliters of phosphate-buffered saline(also known as PBS: 150 mM, NaCl; 100 mM, NaP, pH 7.2) were used forassembling eight microparticle arrays on silicon chips (2.5×2.5 mm) with4,000 microwells on each chip. The following procedures were used:

-   -   (1) Microparticles from PBS were collected in an 1.5 ml        centrifuge tube by centrifugation (14,000 g, 1 minute). Other        collection means may be used.    -   (2) The supernatant was discarded by aspiration using a transfer        pipet.    -   (3) The particles were re-suspended in 5 microliters of 5%        glycerol in 10 mM Tris pH 7.5.    -   (4) The particles were collected from the glycerol solution by        centrifugation. Other collection means may be used.    -   (5) The glycerol solution was aspirated from the particle        pellets.    -   (6) The pellets were re-suspended in 1 microliter of the 5%        glycerol, 10 mM Tris, pH 7.5.    -   (7) Eight silicon chips were placed on a double-sided tap        attached on a microscope slide.    -   (8) A 0.1 microliter volume of the particle suspension was        pipetted onto each of the chips in the area with 4,000        microwells.    -   (9) A cotton applicator was washed with water from a wash        bottle.    -   (10) The wet cotton applicator was blown dry for 30 seconds by        using pressured air. The airflow removes excess water from the        cotton of the applicator. In addition, the air also blows out        some fibers from the surface, which makes the cotton ball more        fluffy.    -   (11) Due to evaporation of the bead suspension in the air and        hygroscopic nature of glycerol in the solution, by the time        steps 9 and 10 were completed (about 1-2 minutes), the water        content in the suspension from step 8 reached equilibrium.        Because of increased viscosity, the droplet became more of a        slurry. To assemble microparticle arrays, the bead slurry was        gently stirred with the tip of the wet cotton applicator in a        circular motion several times. The loose fibers of the cotton        ball ferried the beads into the microwells on the surface (FIG.        3).    -   (12) The particle occupancy of the microwells was examined by        using a fluorescent microscope. If the occupancy is not        satisfactory, step 11 can be repeated.    -   (13) Excess particles were gently wiped away from the chip by        using the cotton applicator. To avoid excess water on the        surface, the cotton applicator was not pressed against the chip.    -   (14) The chip was dried by blowing on the surface of the chip        with compressed nitrogen.    -   (15) The assembled microparticle prepared by this method can be        used for assays or stored in solution at 4° C. for later use.

In this example, the microparticles are suspended in a small amount of5% glycerol, 10 mM Tris pH 7.5 solution for direct deposition ofmicroparticle arrays on the silicon chip. However, while the particlesmay be suspended in other solutions, if LEAPS is used to assemble thebeads, high solution viscosity or ionic concentration may interfere withLEAPS, (e.g., with the assembly of particles on designated areas of asubstrate such as a patterned or illuminated electrode). Accordingly, itis recommended that the ionic concentration of the suspension be about1.0 mM or lower, preferably between about 0.1 mM to 1.0 mM. In addition,it is recommended that the viscosity of the suspension be about 100 cpor less.

In addition, certain salts, such as sodium phosphate and sodiumchloride, may form crystals at the elevated concentrations that occurduring step 11. Such crystals may interfere with bio-molecules on beadsurfaces. Accordingly, they are not recommended for use in beadsuspensions.

Example 4 Direct Deposition

The direct deposition method disclosed in the present application is asimple approach for assembling microparticle arrays on a solid surfaceefficiently. For example, a 0.25 microliter volume of 1% microparticlesolution (10 mg/ml, which corresponds to 168,000 beads), is enough forassembling arrays on a silicon chip containing 4,000 microwells withhigher than 95% occupancy. In other words, it takes about 2% of thebeads in suspension to fill in the microwells at the surface. Inaddition, the assembly process is carried out in water solution withneutral pH, at room temperature. These mild conditions assure that thereactivity of molecules such as DNA, RNA, peptides, and proteins, whenimmobilized on the particles, remains unchanged in the assembly. In thisway, the microparticle arrays assembled using this method are compatiblewith various biochemical assays. Furthermore, the assembly process canbe scaled up from single chip assembly to wafer-scale assembly, and canbe automated to produce large numbers of microparticle arrays.

The direct deposition method is further illustrated by the followingexample. A volume of 2 microliters of a 1% microparticle solution(microparticles approximately 3.5 micrometers in diameter) were added to100 microliters of phosphate-buffered saline (also known as PBS: 150 mM,NaCl; 100 mM, NaP, pH 7.2) to form eight microparticle arrays on siliconchips (2.5×2 5 mm) with 4,000 microwells on each chip. The procedure wasas follows:

-   (1) Microparticles were collected from the PBS in an eppentof tube    by centrifugation (14,000 g, 1 minute). Other collection means may    be used.-   (2) The supernatant was discarded by aspiration using a transfer    pipet-   (3) The particles were re-suspended in 5 microliters of 5% glycerol    in 10 mM Tris pH 7.5.-   (4) The particles were collected from the glycerol solution by    centrifugation. Other collection means can be used.-   (5) The glycerol solution was aspirated from the particle pellets.-   (6) The pellets were re-suspended in 1 microliter of the 5%    glycerol, 10 mM Tris, pH 7.5 solution.-   (7) Eight silicon chips were placed on a double-sided tap attached    to a microscope slide.-   (8) A volume of 0.1 microliter of the particle suspension was    pipetted onto each of the chips in the area with 4,000 microwells.-   (9) The cotton applicator was washed with water from a wash bottle.-   (10) The wet cotton applicator was blown dry for 30 seconds by using    pressurized air. The airflow removes excess water from the cotton    applicator. In addition, the air also blows out some fibers from the    surface, which makes the cotton ball more fluffy.-   (11) Due to evaporation of the bead suspension in the air and the    hygroscopic nature of the glycerol in the solution, by the time    steps 9 and 10 were finished (1-2 minutes), the water content in the    suspension from step 8 reached equilibrium. Because of increased    viscosity, the droplet becomes a slurry. To assembly microparticle    arrays, the bead slurry was gently stirred with the tip of the    moistened cotton applicator in a circular motion several times. The    loose fibers of the cotton ball will ferry the beads into the    microwells on the surface.-   (12) The particle occupancy of the microwells was examined by using    a fluorescent microscope. Step 11 was repeated in cases where the    occupancy was not satisfactory.-   (13) Excess particles were gently wiped away from the chip by using    the cotton applicator. To avoid excess water on the surface, the    cotton applicator was not pressed against the chip.-   (14) After step 13, the assembled microparticle arrays were ready    for assays, or for storage in solution at 4° C. for later use.

For assembling arrays using direct deposition, it is useful to usemicroparticles suspended in small amount of 5% glycerol, 10 mM Tris pH7.5 solution. The use of concentrated glycerol, (i.e. higher than 5%),may increase the viscosity of the bead slurry, and the specific gravityof the solution in the droplet on the chip (step 11). In turn, this maycomprise the assembly efficiency. Although the solution used for thedirect deposition method is not limited to 10 mM Tri, pH 7.5, it shouldbe noted that certain salts, such as sodium phosphate and sodiumchloride, tend to form crystals in elevated concentrations, such as instep 11. The salt crystals not only serve to reduce the occupancy ofmicroparticles in the wells, but also may damage molecules on thesurface during assembly.

It is also recommended to store the assembled chips or wafers comprisingchips in a humid chamber for a short period of time (e.g. 30 min) toallow the beads to settle down in the recesses by gravity before beingused in an assay. Centrifugation of assembled arrays bound to a glassslide may facilitate the settling process. Recommended settings for thecentrifugation are as follows:

-   -   Centrifuge: Sorvall centrifuge model RT6000B    -   Rotor: Sorvall swing bucket model H1000B    -   Speed: 2000 RPM    -   Time: 5 min    -   Operation note: Set the centrifuge at refrigerated mode at 10°        C.        -   Set the brake at off mode        -   Slow ramp up the speed from 0 to 2000 in the first 2 min            followed by centrifugation at 2000 RPM an additional 5 min.        -   Equivalent equipment and settings may be used for this            process.

Viscous immersion media are useful for mounting the chips on the slidefor microscope examination. One example is to use mounting mediacontaining 2.25 M tetrathylammonium chloride, 37.5 mM Tris, pH 8.0, 25%glycerol.

Example 5 Parallel Assembly of Biochip Arrays

The present invention provides methods for parallel assembly of biochiparrays. In this embodiment, the biochip arrays are formed from chipsthat originate from different wafers. A non-limiting example isillustrated by FIG. 9, which shows four different wafers giving rise tofour types of chips: A, B, C, and D. Rows or columns of chips may becombined in any geometry to form an intermediate chip matrix. Inpreferred embodiments, the chips have a regular geometric shape, (forexample, a square or rectangle), and the corresponding intermediate chipmatrix also has a regular geometric shape. Rows or columns are thenextracted from the intermediate chip matrix, such that the rows orcolumns comprise different types of chips. Depending on the application,the mixed rows or columns may contain more than one copy of a certaintype of biochip. The mixed rows and/or columns formed in theseembodiments can be incorporated into biochip arrays for bioassays. Inpreferred embodiments, semiconductor chip-handling equipment is used toassemble the intermediate chip matrix and to extract the mixed rows orcolumns. By using long rows or columns of chips to form the intermediatechip array, it is possible to generate many mixed rows or columnssimultaneously. In this way, it is possible to mass-produce the mixedrows or columns.

Example 6 Biochip Protection by Saccharide Coating

Functionalized beads were assembled on a chip using standard procedures.Following assembly, the chip surface was cleaned, and 2-4 μl of 1%solution of trehalose (alpha-D-glucopyranosyl alpha-D-glucopyranoside, anaturally-occurring, glass-forming disaccharide) in DI water wasdispensed on the chip (surface dimension: 1.75×1.75 mm) and allowed todry under ambient conditions. On drying, a glassy film formed on thesubstrate and encapsulated the assembled beads. Although the film isstable even under high humidity conditions, exposure to liquid waterdissolves the film instantly.

To evaluate the effect of the film formation on the activity of thefunctionalized particles, neutravidin-functionalized particles wereassembled on biochips. Some biochips were passivated with trehalosesolution as described above and subjected to normal ambient conditions,while other biochips were not coated with trehalose solution but insteadstored at 4° C. for 2 weeks. It was found that the bioactivity of thebio-coated chips was similar to that of the non-coated biochips kept at4° C.

Example 7 Hydrogels as Multifunctional Agents in Wafer Cleaning, Storageand Particle Recovery

Agarose hydrogel can be employed as a peeling agent to remove theparticles from a chip in a manner that permits them to be retrievedlater. The hydrogel also can be used as a storage material to preventwafer and particles from dehydration and dust.

Functionalized particles were assembled on a 6 inch wafer comprised ofchips. To clean the particles left on the surface, a 1% agarose solutionat 55° C. (melting point 95° C., gelation temperature 50° C.) was pouredonto the wafer, and kept under ambient conditions or at 4° C. until thegelation occurs. Gels with different thickness, from micrometers tomillimeters, can be produced by using spacers of different thickness.The spacers provide a barrier at the edge of the wafer to prevent theagarose solution from running off the edge. The beads located on thesurface of the wafer, rather than inside the recesses, will be embeddedin the gel. After the solution is completely solidified, the gel film,as well as embedded beads, can be easily peeled off A compressednitrogen stream is then applied immediately to blow dry the small amountof water residue on the surface. In this way, the wafer surface remainsclean.

To assess the effect of the peeling procedure on the occupancy, as wellas the effect of the agarose gel film on the activity of thefunctionalized particles, the particles were assembled on the chips andthe chips were then subjected to decoding analysis and extension assays.FIG. 12 showed that the peeling procedures did not decrease theoccupancy (i.e. no particles were pulled out of recesses). It is believethat the viscosity of the gel solution plays a role in maintaining theparticles inside of holes. With higher gel solution viscosities,tendency of solution to go into the recess before gelation decreases, sothere is a lower probability that the occupancy is affected. An SSPon-chip assay indicated that the signal and CV were comparable (FIG. 13ab), indicating that the gel does not affect the assay sensitivity.

Agarose gel is a thermo-reversible physically crosslinked hydrogel. Forthe purpose of particle recovery, agarose with ultra low melting point(m.p.<50° C., gelation temperature, 8-17° C.) should be used.Subsequently, the agarose gel can be re-melted at 50-55° C., and theembedded particles can be retrieved. The biological activities ofbiomolecules on the particles are retained under these conditions.

Such hydrophilic hydrogels not only can be used as a peeling agent, butalso as a storage agent to prevent particles/wafers from dehydration,dust and physical damage during the storage and shipping.

Example 8 Polymer Coating

A small batch of cleaned individual chips [about 5 to 20 in number] wereplaced in a small TEFLON™ (polytetrafluoroethylene) container (volume ˜5ml) filled with 1 ml of a 1%

(1 mg/ml) solution of polyallylamine hydrochloride (Mw˜15,000) or a 0.1%polylysine solution (Sigma Aldrich). The chips were incubated for 1-2 hrwith gentle shaking at room temperature. Afterwards, they were removedfrom the polymer solution and dried for ˜1 hr in the temperature rangeof 50-70° C. This treatment usually leaves behind a thick and unevencoating film of the polymer on the surface of the chip. These modifiedchips were used for assembling beads using standard protocols. Thesurface cleaning step at the end of the assembly process removed most ofthe excess polymer along with the excess beads. The presence of thepolymer coating improved the adhesion of the beads to the chip surfaceand the retention of the beads in the recesses were considerablyimproved after such a treatment.

Example 9 Biochip Packaging to Form Multi-Chip Carriers for BiologicalAssays and Addition to Bead Arrays and Methods of Preparation Thereof

The type of packaging chosen for a particular biochip depends on theapplication. Usually, one or more biochips are affixed on a chip carrierfor convenience. The carriers can be as simple as glass slides, or theycan be complicated cartridges with fluidic handling, temperaturecontrol, signal recording, and other functions. The biochips can bebonded to the carrier permanently by glue or reversibly bonded byvarious means such as magnetic or mechanical forces.

Example 9A A Multi-Chip Carrier Made from a Glass Slide

To prepare the slide as a carrier, a TEFLON™ (polytetrafluoroethylene)coating is applied in such a manner as to leave circular openings orwells (i.e., areas of glass without any TEFLON™ covering). Each well isa circle with 6.5 mm in diameter. One or more chips can be bonded to theglass surface within a well. A typical glass slide is 25×75 mm and 1 mmthick, with a 2×5 array of wells. With typical chip sizes of 1.75×1.75mm, up to four chips can be bonded to the glass surface in each well.Each chip in the same well can have distinct bead groups that wereassembled prior to bonding to the carrier. For example, if each chip hasan array containing 39 types of bead groups, a well with 4 distinctchips would have a total of 4×39=156 types of beads. On the other hand,for larger chips (e.g., a 4.5×4.5 mm square) an entire well is occupiedby a single chip. For the well dimensions described herein, each wellcan hold up to 40 μl of liquid (usually an aqueous solution). Typically,a 20 μl volume of sample solution is added to each well for biologicalreactions, such that each chip is totally covered by the samplesolution. Because the TEFLON™ (polytetrafluoroethylene) coating outsidethe wells is hydrophobic, the aqueous samples do not spill out. Theformat of a carrier slide can be designed to fit certain applications.For example, a single row of 8 wells on a slide can be used to analyze 8samples. Furthermore, a 4×8 array of wells can be used to analyze 32samples. Similarly, more wells (e.g., 96, 384, and 1536) can be arrangedon a single slide to analyze more samples.

Example 9B Mobile Chip Carrier

In certain embodiments of a mobile chip carrier, chips are bonded to asubstrate such as glass, stainless steel, plastic materials,semiconductors, or ceramic materials. The whole carrier unit is movableand can be transported during processing to expose the chips todifferent reaction media, such as reaction chambers, washing chambers,and signal reading stages. (See FIG. 14 for an embodiment).

In other embodiments, the mobile chip carrier comprises a chamber orchambers in which the chips are bonded. By housing the chips inside themobile chip carrier, contamination during transport can be minimized. Incertain embodiments, the chamber(s) of the mobile chip carrier alsoserve as a processing environment. Reactive gases or liquid solutionsfor various purposes, such as performing a bioassay or cleaning thechips, may be admitted into the mobile chip carrier and subsequentlyevacuated, if desired. Additionally, the mobile chip carrier may possessmeans for changing the thermodynamic properties of the chamber, such asthe chamber pressure or temperature.

Example 10 Assembly of Encoded Chip Arrays by Random Tiling

A multiplicity of chips can be produced by the assembly of randomencoded arrays of probes that are displayed on beads. Each chip mayinclude one or more random encoded bead arrays and may be cut from auniquely identified wafer. As illustrated in FIG. 15 (a, b, c and d), arandom encoded array of chips may be produced in a tiling process. Thisprocess may be facilitated by a choosing an appropriate chip shape inorder to facilitate alignment and to maximize interlocking placement todecrease the array-to-array distance.

What is claimed is:
 1. A biochip comprising a semiconductor wafersubstrate having a multiplicity of bead arrays within delineatedcompartments positioned thereon and having separating boundaries betweensaid delineated compartments, wherein each bead array comprises a set ofbiofunctionalized beads and wherein the biofunctionalized beads areoptically encoded such that bead subpopulations having differentbiomolecules attached thereto are distinguishable by optical analysis;and wherein each of the delineated compartments comprises recessstructures for housing the biofunctionalized beads, wherein thestructures each comprise a recess surface opening and a recess bottom,and wherein the recess surface opening comprises a smaller diameter thanthe recess bottom.
 2. The biochip of claim 1, wherein the separatingboundaries comprise scribe lines.
 3. The biochip of claim 1, wherein thebiofunctionalized beads comprise one or more biomolecules selected fromthe group consisting of oligonucleotides, nucleic acid fragments,proteins, oligopeptides, ligands, receptors, antigens, antibodies,individual members of biological binding pairs, and combinationsthereof.
 4. The biochip of claim 1, wherein the structures aresubstantially occupied by biofunctionalized beads.
 5. The biochip ofclaim 1, wherein the structures are each sized so as to accommodate onebead.
 6. The biochip of claim 1, wherein the structures are each sizedso as to accommodate multiple beads.
 7. The biochip of claim 1 whereinthe structures comprise triangular, rectangular, pentagonal, hexagonal,circular shapes or a combination thereof.
 8. The biochip of claim 1,wherein the structures have a depth of about 0.5 to 1.5 times thediameter of the beads.
 9. The biochip of claim 1, wherein each beadarray comprises the same set of biofunctionalized beads.
 10. The biochipclaim 1, wherein the multiplicity of bead arrays comprises at least twodifferent sets of biofunctionalized beads.
 11. The biochip of claim 1,wherein each delineated compartment is associated with a decodable tagthat identifies the semiconductor wafer substrate.
 12. The biochip ofclaim 1, wherein the biofunctionalized beads are affixed to thesubstrate by covalent bonds, van der Waals forces, electrostatic forces,gravitational forces, magnetic forces, or a combination thereof.
 13. Thebiochip of claim 1, wherein the biofunctionalized beads compriseplastic, ceramic, or glass beads or a combination thereof.
 14. Thebiochip of claim 1, wherein the biofunctionalized beads have diametersof from about 0.2 micron to about 200 microns.
 15. The biochip of claim1, wherein the optical analysis comprises analysis of one or more ofexcitation wavelength, emission wavelength, excited-state lifetime, andemission intensity.
 16. The biochip of claim 1, further comprising aremovable protective coating covering the multiplicity of bead arrays.17. The biochip of claim 16, wherein the removable protective coatingcomprises an inert, non-reducing sugar or a hydrogel.
 18. A plurality ofchips prepared by singulation of the biochip of claim 1 along theseparating boundaries.